Project Summary The ultimate goal of this project is to enable the use of engineered cell therapies to safely and effectively treat conditions ranging from cancer, to autoimmune disease, to those requiring regenerative medicine. Engineered cell therapies represent an exciting frontier in medicine, and early successes in the field of cancer treatment have demonstrated the transformative potential of this approach, enabling the treatment of patients for whom no existing therapy was effective. However, fully realizing the promise of engineered cell therapies will require technologies and tools that enable bioengineers to efficiently design, build, and evaluate customized cellular functions that meet specific clinical needs. While the field of mammalian synthetic biology has made impressive strides toward this goal, translating basic science to the clinic imposes a new set of design and engineering challenges. In particular, new experimental technologies and computational tools are needed to design cell therapies in a way that leads to robust performance?the successful execution of a therapeutic program despite inevitable biological variability. To meet this need, this team will develop an integrated suite of new experimental reagents, new computational tools, and new conceptual understanding to accelerate the implementation of design-driven medicine by enabling bioengineers to program cells to sense, evaluate, and respond to their environment in novel, useful, and reliable ways. The team recently developed a synthetic biology technology called MESA receptor proteins, which enable one to ?rewire? how a cell senses features of host physiology. This project comprises a crucial bridge from an early demonstration of a promising strategy to the development of a true technology platform that may be readily applied by the bioengineering community to design and construct novel cell therapies. The goals of this project are informed by the team's substantial experience in engineered receptor technologies, and this project addresses general challenges in mammalian synthetic biology. The first Aim is to develop strategies for engineering cellular sensing functions that perform robustly across inevitable biological variation. This aim comprises computational model-guided design of proteins and genetic components to make cellular sensing functions more useful for bioengineering. This work will include a comparison of MESA with other engineered sensing platforms. The second Aim is to develop a library of novel MESA biosensors that respond to physiologically relevant cues. Outcomes of this aim will include a better understanding of how to build biosensors, as well as a panel of reagents that enable bioengineers to immediately employ this technology for therapeutic applications. The third Aim is to evaluate and develop strategies for implementing engineered biosensing functions in a wide range of cell types, including both stable cell lines and primary cells, with comparisons across translationally-relevant gene delivery platforms.